The present invention relates to the use of 12-methyltetradecanoic acid which can kill tumor cells by inducing cell apoptosis related to inhibition of 5-lipoxygenase metabolites and 12-lipoxygenase metabolites or their related activities in various mammalian cancers. In particular, the present invention relates to the methods using 12-methyltetradecanoic acid (12-MTA) alone or in combination with other anti-cancer compounds that are targeted to inhibit cancer progression in a mammal by inducing tumor cell apoptosis.
Normal tissue homeostasis is maintained by balanced cell proliferation and cell death, which occurs most frequently in the form of apoptosis or programmed cell death. Tumor cells differ significantly from their normal counterparts with respect to the control of cell growth and proliferation. Most tumor cells demonstrate a self-dominant growth pattern either due to their abnormal response to environmental stimuli (hormones, growth factors, cytokines, etc.) or due to an autonomous nature of growth (i.e., autocrine stimulation). Tumor cells also demonstrate abnormal apoptotic responses. Many factors have been shown to regulate apoptosis, including (i) growth factors and growth factor receptors such as retinoid acid, interleukin-3, stem cell factor, interferon-xcex3, erythropoietin, NGF/NGF (nerve growth factor) receptor, TNF-xcex1/Fas (tumor necrosis factor-xcex1), steel factor/Kit receptor, TGF-xcex2/TGF (transforming growth factor) receptor, insulin, EGF/EGFR (epidermal growth factor), IGF-1/IGF (insulin-like growth factor) receptor, and PDGF/PDGF (platelet-derived growth factor receptor); (ii) intracellular signal transducers such as protein kinase C, PI-3 (phosphoinositol-3) kinase, Ras and GTPase, PLC-xcex3 (phospholipase C-xcex3), tyrosine kinases and protein phosphatases, lipid signaling molecules such as eicosanoids, sphingosine, ceramide, and Ca2+; (iii) cell cycle regulators exemplified by Cdc-2 and E2F-1; (iv) reactive oxygen species or other free radicals; (v) extracellular matrix regulators/cell adhesion molecules (extracellular matrix proteins such as fibronectin and transmembrane integrin receptors); and (vi) specific endonucleases such as Ca2+- and Mg2+-dependent DNase and cytoplasmic proteases typified by ICE (interleukin 1xcex2-converting enzyme) family. Many of these regulators have been associated with various human malignancies and apoptosis. For example, studies on human tumors including neuroblastoma, glioma, lymphoma, breast carcinoma, colorectal adenocarcinoma, melanoma and gastrointestinal malignancies have demonstrated an overall positive correlation between increased expression of Bcl-2 (or Bcl-XL) or decreased expression of Bax and uncontrolled tumor cell growth, and, in some cases, with tumor progression and a poor prognosis of cancer patients. Another example is p53, a phosphoprotein known to modulate gene transcription, police cell cycle checkpoints, control DNA replication and repair, and maintain genomic stability. Wild type p53 also positively regulates apoptosis. p53 gene mutations have been linked to attenuated apoptosis in multiple cancers represented by Wilms"" tumor, colon cancer, cervical carcinoma and breast cancer. Since apoptosis plays a critical role in multiple steps (transformation, progression and survival of metastases) of tumorigenesis as well as in tumor cells"" response to chemotherapeutic drugs or radiation therapy, many chemoprevention and therapeutic regimens attempting to manipulate apoptotic process have been proposed to aid in the clinical treatment of cancer patients (Fesus, L., et al., J. Cell Biochem. 22:151-161 (1995); Lotan, R., J. Natl. Cancer Inst. 87:1655-1657 (1995); van Zandwijk, N., J. Cell Biochem. 22:24-32 (1995)).
Arachidonic acid (AA) is an essential component of the cell membrane phospholipids. AA released through the action of phospholipase A2 is metabolized via three major biochemical pathways: (i) the cyclooxygenase (COX) pathway leading to the generation of prostaglandins, prostacyclin, and thromboxane; (ii) the lipoxygenase (LOX) pathway giving rise to various hydroperoxy (HPETEs) and hydroxy (HETEs) fatty acids as well as leukotrienes; and (iii) the P450-dependent epoxygenase pathway generating EETs. Mammalian LOX display varying degrees of substrate specificity for insertion of molecular oxygen into arachidonic acid at carbon positions 5, 12, and 15. The enzymes, based on the abundance of the majority products have thus been termed 5, 12, and 15 lipoxygenases, respectively. The 12-LOX catalyzes the transformation of AA into 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) and its 12(S)-hydroxy derivatives, i.e., 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Three types of mammalian 12-LOX enzymes have so far been reported. The first is human platelet-type 12-lipoxygenase expressed normally in platelets, HEL (human erythroleukemia) cells, and umbilical vein endothelial cells (Funk, C. D., et al., Proc. Natl. Acad. Sci. 87:5638-5642 (1990); Funk, C. D., et al., Proc. Natl. Acad. Sci. 89:3962-3966 (1992)). Platelet-type 12-LOX metabolizes only AA (but not C-18 fatty acids such as linoleic acid) to form exclusively 12(S)-HETE (Funk, C. D., et al., Proc. Natl. Acad. Sci. 87:5638-5642 (1990) Marnett, L. J., et al., Adv. Prostaglandin Thromboxane Leukotriene Res. 21:895-900 (1990)). The second is porcine leukocyte-type 12-LOX which metabolizes both AA and linoleic acid thus generating 12(S)-HETE as well as small amounts of 15(S)-HETE (Hada, T., et al., Biochim. Biophys. Acta 1083-1087 (1991)). The third type of 12-LOX (sometimes termed epithelial 12-lipoxygenase) has been isolated from bovine tracheal epithelial cells (De Marzo, N., et al., J. Physiol. 262:L198-L207 (1992)); rat brain (Watanabe, S., et al., Eur. J. Biochem. 212:605-612 (1993)), and murine macrophages (Freier-Moar, J., et al., Biochim. Biophys. Acta., 1254:112-116 (1995)), which shares more homology with 15-LOX and leukocyte-type 12-LOX than with platelet-type 12-LOX. This type of 12-LOX, like reticulocyte 15-LOX and leukocyte-type 12-LOX, catalyzes the formation of both 12(S)-HETE and 15(S)-HETE.
The role of AA metabolites in regulating cell proliferation has been recognized for more than two decades. Numerous studies have demonstrated a strong positive correlation between growth factorxe2x80x94(EGF, insulin, PDGF, etc.) promoted cell proliferation and generation of various COX products, primarily prostaglandins (Skouteris, G. G., et al., Biochem. Biophys. Res. Commun., 178:1240-1246 (1991); Nolan, R. D., et al., Mol. Pharmacol. 33:650-656 (1988); Smith, D. L., et al., Prostaglandins Leukotrienes Med., 16:1-10 (1984)). Similarly, it has been found that various eicosanoids derived from LOX pathways as well as epoxygenase pathways of AA metabolism play an essential role in mediating the growth factor-stimulated normal cell and tumor cell growth. Examples include 15-HETE as a mitogenic regulator of T-lymphocyte (Bailey, J. M., et al., Cell Immunol., 67:112-120 (1982)), 12-HETE and LTB.sub.4 as growth stimulators of epidermal cells (Chan, C., et al., J. Invest. Dermatol., 85:333-334 (1985)), 12-HETE stimulation of keratinocyte DNA synthesis (Kragballe, K., et al., Arch. Dermatol. Res., 278:449-453 (1986)), 15-/12-HETEs as mediators of insulin and EGF-stimulated mammary epithelial cell proliferation (Bandyopadhyay, G. K., et al., J. Biol. Chem., 263:7567-7573 (1988)) and as synergistic effectors of bFGFxe2x80x94(basic fibroblast growth factor) and PDGF-regulated growth of vascular endothelial cells and smooth muscle cells (Yamaja Setty, B. N., et al., J. Biol. Chem., 262:17613-17622 (1987); Dethlefsen, S. M., et al., Exp. Cell Res., 212:262-273 (1994)), 12(S)-HETE as a regulator of EGF- and insulin-stimulated DNA synthesis and protooncogene expression in lens epithelial cells (Lysz, T. W., et al., Cell Growth and Differ., 5:1069-1076 (1994)) and as the mediator of angiotensin II-induced aldosterone synthesis in adrenal glomerulosa cells (Nadler, R. D., et al., J. Clin. Invest., 80:1763-1769 (1987)).
Early work demonstrated an important function for prostacyclin (PGI2) and thromboxane (TxA2), two major cyclooxygenase (COX) products of AA metabolism derived primarily from vascular endothelial cells and platelets, respectively, in regulating the hematogenous spreading of malignant tumor cells, reviewed in Schneider et al., Cancer metastasis Rev. 13:349-364 (1994). Later, systematic in vitro and in vivo studies have led to the discovery that many LOX metabolites also play a key role in modulating the phenotypic properties of tumor cells as well as tumor cell-vasculature interactions (Reviewed in Cancer Metastasis Rev. 11:353-375 (1992); Prominently, a small hydroxy fatty acid molecule derived from the LOX pathway of AA metabolism, i.e., 12(S)-HETE, has been observed to possess a wide-spectrum of biological activities including, among others, inducing platelet aggregation, stimulating insulin secretion, suppressing renin production, chemoattracting leukocytes, facilitating macrophage adhesion, inhibiting prostacyclin biosynthesis by vascular endothelial cells (Spector, A. A., et al., Prog. Lipid Res. 27:271-323 (1988); Sekiya, K., et al., Biochem. Biophys. Res. Commun. 105:1090-1095 (1982), modulating tumor cell interactions with extracellular matrix, promoting tumor cell motility, facilitating tumor cell release of proteolytic enzyme cathepsin B, reorganizing tumor cell cytoskeleton, promoting tumor cell adhesion on endothelial cells via upregulating integrin expression on tumor cells and/or endothelial cells, and inducing endothelial cell retraction thus enhancing tumor cell extravasation from the vasculature (reviewed in Seminar Thromb. Hemost. 18:390-413 (1992); Cancer Metastasis Rev., 13:365-396 (1994); Annals New York Acad. Sci., 744:199-215 (1994); Invasion Metastasis 14:109-122 (1995). Significant progress has been made in delineating the molecular mechanisms of the 12(S)-HETE effects. 12(S)-HETE, possibly through binding to a cell surface receptor(s), triggers phosphoinositol lipid hydrolysis (Liu, B., et al., Proc. Natl. Acad. Sci. 92:9323-9327 (1995)) leading to the intracellular activation of protein kinase C (PKC; Liu, B., et al., Cell Regul. 2:1045-1055 (1992)) and/or protein tyrosine kinase (PTK; Tang, D. G., et al., J. Cell. Physiol. 165:291-306 (1995c)). The interactions of these phosphorylated protein kinases with various intracellular molecular targets (e.g., cytoskeletal proteins, adhesion molecules, signaling molecules, etc.) largely explain the versatility of the 12(S)-HETE effects.
Both COX and LOX products of AA metabolism may also be involved in modulating tumor cell survival and apoptosis. Thus, many prostaglandins such as PGE2 (prostaglandin E2) (Brown, D. M., et al., Clin. Immunol. Immunopathol. 63:221-229 (1992)), PGA2 and xcex9412  -PGJ2 (Kim, I-K, et al., FEBS Lett. 312:209-214 (1993)) have been shown to induce apoptosis of leukemia or lymphoma cells as well as solid tumor cells. Similarly, TxA2 induces apoptotic cell death of immature thymocytes by binding to the cell surface TxA2 receptors (Ushikubi, F., et al., J. Exp. Med. 178:1825-1830 (1993)). Interestingly, PGE2 also has been reported to protect cells from apoptosis induction (Goetzel, E. J., et al., J. Immunol. 154:1041-1047 (1995)). On the other hand, various COX inhibitors (NSAID) (Non-steroidal anti-inflammation drugs) have been consistently demonstrated to trigger apoptosis of cultured cells. Thus, indomethacin, sulindac sulfide and sulfone inhibit colon carcinoma cell (HT-29) growth by inducing apoptosis (Shiff, S. J., et al., J. Clin. Invest. 96:491-503 (1995); Piazza, G. A., et al., Cancer Res. 55:3110-3116 (1995); Shiff, S. J., et al., Exp. Cell Res. 222:179-188 (1996)). Likewise, multiple NSAIDs including diflunisal, indomethacin, acemethacin, diclofenac, mefenamic acid, flufenamic acid, niflumic acid, ibuprofen, and carprofen cause apoptosis in chicken embryo fibroblasts (Lu, X., et al., Proc. Natl. Acad. Sci. 92:7961-7965 (1995)). These observations suggest that the COX/COX metabolites may play a dual role in regulating cell survival and apoptosis. Under certain circumstances the COX products (e.g., PGE2 and cyclopentenone prostaglandins) can either directly trigger cell death or mediate apoptosis induced by, e.g., TNF-xcex1 (Larrick, J. W., and S. C. Wright, FASEB J. 4:3215-3223 (1990)). In a different scenario, the COX activity/function are critical for cell survival since inhibition with various inhibitors leads to cell death (see above). Consistent with this, overexpression of COX-2 has been observed to confer resistance in rat intestinal epithelial cells to apoptosis induction by butyrate (Tsujii, M., and R. DuBois, Cell 83:493-501 (1995)).
Similar to the COX system, some LOXs and their products may also play a dual regulatory role in cell survival and apoptosis. Exogenous lipid hydroperoxides such as 15-HPETE induces HIV-infected human T cells (Sandstrom, et al., J. Biol. Chem. 269:798-802 (1994)) due to their inability to convert 15-HPETE to 15-HETE owing to a reduction in the glutathione peroxidase activity, LOX metabolites have been proposed as the actual mediators of TNFxcex1-induced apoptosis of multiple cells since LOX inhibitors such as ETYA and NDGA could inhibit its cytotoxicity (Chang, D. J., et al., Biochem. Biophys. Res. Commun. 188:538-546 (1992); O""Donell, V. B., et al., Biochem. J. 310:133-141 (1995)). On the other hand, 5-LOX inhibitors can cause apoptosis of human leukemia blast cells (Anderson, K. M., et al., Prosta. Leuko. Essent. Fat. Acids 48:323-326 (1993)) and interruption of 5-LOX-mediated growth factor signaling inhibits the growth of lung cancer cells due to apoptosis induction (Avis, I. M., et al., J. Clin. Invest. 97:806-813 (1996)), suggesting that in some cells the 5-LOX pathway may function as a critical survival factor.
Inflammation inhibition via inhibition of lipoxygenases in mammals is also an aspect of the background of the present invention. Lipoxygenases are involved in several mammalian inflammatory pathologies such as psoriasis, asthma, atopic dermatitis, arthritis, Crohn""s Disease, irritable bowel syndrome and others.
Japanese patent application JP 7002661A describes 12-MTA as being an inhibitor of myeloperoxidase secretion from polymorphonuclear leukocytes. The patent application does not describe 12-MTA as an inhibitor of lipoxygenase nor of cancer cell growth, or as an inducer of apoptosis in malignant cancer cells.
It has been shown by Collin (PCT/US99/01179) that lipids derived from sea cucumber, especially of the class Cucumaria frondosa, inhibit 5- and 12-lipoxygenase. Related to this lipoxygenase inhibition is the inhibition of inflammation in mammals, both systemically and topically. Surprisingly, it was found by the present inventors that one compound isolated from Cucumaria frondosa, 12-MTA, is a dramatic inhibitor of lipoxygenases and a dramatic inhibitor of various cancer cell proliferation in vitro.
Inhibition of various lipoxygenase activities in cancer victims or for prevention of cancers, through administration of lipoxygenase inhibitors is a promising therapy for humans and animals. Currently, no lipoxygenase inhibitors have been approved for general anti-cancer therapies.
It is therefore an object of the present invention to provide a method for the inhibition of cancer proliferation in humans and other animals by the administration of 12-MTA or 12-MTA in combination with other nutritional or anti-cancer compounds, either topically, orally, intraperitoneally or other means known to the medical arts.
It is as additional object of the present invention to provide a composition of matter consisting of 12-MTA derived from sea cucumber, preferably comprising 60% or more 12-MTA, between 10 and 20% palmitoleic acid, between 10 and 30% eicosapentaenoic acid (EPA), and less than 10% of other fatty acids, including myristic acid (14:0) and oleic acid (18:1), useful as an anti-inflammatory therapeutic product. Such a product is useful in inhibiting inflammation in a mammal by reducing the expression of lipoxygenase activities, especially in conjunction with other anti-inflammatory compounds which inhibit the cyclooxygenase cascade in vivo.
It is an additional object of the present invention that 12-MTA can be complexed with currently approved anti-cancer agents and that such a complexed compound will render some current drugs more efficacious. For example, gemcitabine esters or amides in which the 3xe2x80x2 and/or 5xe2x80x2 OH group and/or the N4-amino group are derivatised with 12-MTA, can be made by those skilled in the arts, thus making the complex more useful as an anti-cancer agent. Other anti-cancer compounds such as cyclophosphamide (alkylating agent), 5-fluorouracil (antimetabolite), etoposide (semisynthetic podophyllotoxin agent) and vincristine (vinca alkaloid) may be compounded with 12-MTA or administered separately by being spaced out over time in dosages that provide an effective amount of each synergistically.
The present invention also relates to combinations of 12-MTA with paclitaxel (Taxol(copyright), Bristol Myers Squibb), docetaxel (Taxotere(copyright). Rhone-Poulenc Rorer) and their analogues and substances which are therapeutically useful in the treatment of neoplastic diseases.
Taxol(copyright), Taxotere(copyright) and their analogues, which possess noteworthy antitumor and antileukemic properties, are especially useful in the treatment of cancers of the ovary, breast or lung.
The preparation of Taxol(copyright), Taxotere(copyright) and their derivatives form the subject, for example, of European Patents EP 0,253,738 and EP 0,253,739 and International Application PCT WO 92/09,589.
Generally, the doses used, which depend on factors distinctive to the subject to be treated, are between 1 and 10 mg/kg administered intraperitoneally or between 1 and 3 mg/kg administered intravenously.
Among substances which may be used in association or in combination with 12-MTA are: Taxol(copyright), Taxotere(copyright) or their analogues, alkylating agents such as cyclophosphamide, isosfamide, melphalan, hexamethylmelamine, thiotepa or dacarbazine, antimetabolites such as pyrimidine analogues, for instance 5-fluorouracil and cytarabine or its analogues such as 2-fluorodeoxycytidine, or folic acid analogues such as methotrexate, idatrexate or trimetrexate, spindle poisons including vinca alkaloids such as vinblastine or vincristine or their synthetic analogues such as navelbine, or estramustine or taxoids, epidophylloptoxins such as etoposide or teniposide, antibiotics such as daunorubicine, doxorubicin, bleomycin or mitomycin, enzymes such as L-asparaginase, topoisomerase inhibitors such as camptothecin derivatives chosen from CPT-11 and topotecan or pyridobenzoindole derivatives, and various agents such as procarbazine, mitoxantrone, platinum coordination complexes such as cisplatin or carboplatin, and biological response modifiers or growth factor inhibitors such as interferons or interleukins.
It is also an object of the present invention to induce apoptosis of tumor cells in a mammal using 12-MTA and/or 12-MTA in combinations with other anti-cancer agents. Further, it is an object of the present invention to provide methods which can be effective in treating tumors. These and other objects will become increasingly apparent by reference to the following description and the drawings.